Suppressive action of nesfatin-1 and nesfatin-1-like peptide on cortisol synthesis in human adrenal cortex cells

Nucleobindin-derived peptides, nesfatin-1 [NESF-1] and nesfatin-1-like-peptide [NLP] have diverse roles in endocrine and metabolic regulation. While both peptides showed a stimulatory effect on the synthesis of proopiomelanocortin (POMC), the adrenocorticotropic hormone (ACTH) precursor in mouse corticotrophs, whether NESF-1 and NLP have any direct effect on glucocorticoid [GC] synthesis in the adrenal cortex remains unknown. The main aim of this study was to determine if NESF-1 and/or NLP act directly on adrenal cortex cells to regulate cortisol synthesis in vitro. Whether NLP injection affects stress-hormone gene expression in the adrenal gland and pituitary in vivo in mice was also assessed. In addition, cortisol synthetic pathway in Nucb1 knockout mice was studied. Human adrenal cortical [H295R] cells showed immunoreactivity for both NUCB1/NLP and NUCB2/NESF-1. NLP and NESF-1 decreased the abundance of steroidogenic enzyme mRNAs, and cortisol synthesis and release through the AC/PKA/CREB pathway in H295R cells. Similarly, intraperitoneal injection of NLP in mice decreased the expression of enzymes involved in glucocorticoid (GC) synthesis in the adrenal gland while increasing the expression of Pomc, Pcsk1 and Crhr1 in the pituitary. Moreover, the melanocortin 2 receptor (Mc2r) mRNA level was enhanced in the adrenal gland samples of NLP injected mice. However, the global genetic disruption in Nucb1 did not affect most steroidogenic enzyme mRNAs, and Pomc, Pcsk2 and Crhr1 mRNAs in mice adrenal gland and pituitary gland, respectively. Collectively, these data provide the first evidence for a direct inhibition of cortisol synthesis and secretion by NLP and NESF-1. NUCB peptides might still elicit a net stimulatory effect on GC synthesis and secretion through their positive effects on ACTH-MC2R pathway in the pituitary.


Intraperitoneal [IP] injection of NLP modulated the expression of HPA axis-related enzymes, hormones, receptors and endogenous Nucb at mRNA level in mice
IP injection of NLP at 100 µg/kg decreased the expression of steroidogenic enzymes [Star, Cyp21, Cyp11b1 and Cyp11b2] as well as Nucb1 and Nucb2 mRNAs in the adrenal gland (Fig. 6).In contrast, NLP injection enhanced the Mc2r mRNA in the adrenal gland of these mice.Furthermore, NLP injection enhanced the expression of Pomc, Pcsk1 and Crhr1 (Fig. 7A-D) but decreased Nucb1 and Nucb2 mRNAs in mice pituitary (Fig. 7E,F).

Disruption of Nucb1 caused minimal effects to the expression of steroidogenic enzyme mRNAs in the adrenal gland and Pomc, Pcsk2 and Crhr1 in pituitary
The abundance of steroidogenic enzyme mRNAs in the adrenal gland, and HPA-related mRNAs in the pituitary gland of Nucb1 KO mice are shown in Tables 1, 2. The genetic disruption in Nucb1 did not affect the steroidogenic enzyme mRNA abundance in male and female mice, except for the downregulation of Hsd3β in female Nucb1 KO mice (Table 1).Moreover, the expression of Pcsk1 in adrenal gland were significantly lower in male Nucb1 Vol.:(0123456789)
Mc2r mRNA in the adrenal gland (Table 1) and Nucb2 mRNA in the pituitary were unaffected by the disruption of Nucb1 in mice of both sexes (Table 2).

Discussion
This research aimed to determine whether NUCB1/NLP and NUCB2/NESF-1 exist in a human adrenal cortex cell line, and studied if they act directly on steroidogenic enzymes to regulate cortisol synthesis.NUCB2/NESF-1-like immunoreactivity was detected in the cytoplasm and nucleus of human adrenal cortex cells.NUCB2/ NESF-1 immunoreactivity was found in the cytoplasm murine neurons 1,21 and gastric cells 22 and rat brain 23 and Leydig cells 24 , and the human brain 25 .In another study, NUCB2/NESF immunoreactivity was observed H295R cell cytoplasm 26 .This agrees with our results showing cytoplasmic staining of NUCB2/NESF in the same cells.We also NUCB2/NESF immunoreactivity in the nucleus of H295R cells.Previously, NUCB2 immunoreactivity was detected in the nucleus of endothelial cells of mouse lung 27 .Given that NUCBs are DNA binding proteins 1 , the staining found in the nucleus is reflective of that role.NUCB1/NLP-like immunoreactivity was reported in the central nervous system and peripheral tissues, including endocrine cells of the anterior pituitary of rats and mice 1,28 , mouse insulinoma MIN6 cell line and mouse pancreatic beta cells 17 as well as testis, ovary, and head   kidney interrenal cells 2 and pituitary of goldfish 18 .The results reported here show that human adrenal cortex cells express NUCB peptides and possibly other bioactive peptides residing within it, which may act directly on these cells to regulate the steroidogenesis pathway and cortisol synthesis.This suggests that the adrenal gland is likely another tissue source of circulating NUCB and peptides processed from it.The role of locally produced NUCB peptides warrants further consideration.
Immediately after the discovery of NESF-1 29 and its role in metabolic regulation, several studies have examined its implication in anxiety and stress hormone synthesis in vitro and in vivo 30 .NESF-1 stimulates ACTH secretion from goldfish dispersed pituitary cells in vitro 2 .NESF-1 and NLP also stimulate the synthesis of CRH and ACTH in the hypothalamus and anterior pituitary 1,2,12 .However, in the current study, the synthesis and release of cortisol was adversely influenced by NESF-1 and NLP.Cortisol is a catabolic hormone that stimulates      Table 2. HPA axis-related and endogenous Nucb2 mRNAs in the pituitary of wildtype (WT) and Nucb1 KO mice (n = 6 samples or mice/group).No change, an increase, or a decrease in the knockout male or female mice when compared to their respective control groups is denoted in the tables.www.nature.com/scientificreports/oxidation of lipids and decreases the synthesis of lipids and carbohydrates to create a surge in available energy.CRH-stimulated cortisol in humans stimulates the consumption of food 8 .Next, we studied whether NESF-1 and NLP affect steroidogenic enzymes involved in human cortisol synthesis in the adrenal gland.In agreement with our in vitro synthesis and secretory studies described above, both NLP and NESF-1 decreased the expression of most steroidogenic enzymes involved in cortisol synthesis at 12 h post-incubation.Meanwhile, ACTH, as a positive control, increased the expression of these enzymes.These results were complemented by the decreased cellular cortisol content observed in NLP and NESF-1 treated cells and increased cortisol content found in ACTH treated cells at 24 h post-incubation.Another study showed that NESF-1 inhibits the growth of and triggers the apoptosis of H295R cells through BAX, BCL-XL and BCL-2 genes as well as extracellular signal-regulated kinase (ERK1/2), p38 and Jun N-terminal protein kinase (JNK1/2) signalling cascades.These effects of NESF-1 possibly contributed to the suppression of cortisol secretion 26 .We did not see any cell death in our in vitro studies.Since the doses tested and durations of incubation in this research and the above discussed study are different, a direct comparison of results from these two studies are not possible.
In goldfish, ICV and IP administration of NESF-1 increased cortisol secretion and crh mRNA level in vivo and ACTH secretion in vitro 2 .ICV administration of NESF-1 enhanced the secretion of ACTH and corticosterone in rats 12 .In these studies, the net stimulatory effect of NESF-1 on cortisol release seems to be through the ACTH-dependant pathway.The results of this study, although solely using a human cell line, show that NLP and NESF-1 could act directly on the adrenal cortex to inhibit cortisol synthesis and secretion.This effect is ACTH independent.However, as reported in previous studies, both NESF-1 and NLP stimulated POMC synthesis in mouse corticotrophs 1 and NESF-1 stimulated ACTH secretion in goldfish pituitary cells 2 .Therefore, this positive effect of NES-1 and NLP on ACTH synthesis and secretion likely negates any adverse effect these peptides have on cortisol synthesis and secretion in vivo.This is possibly a reason why cortisol was found elevated in vivo after NESF-1 treatment.This comparison has limitations as route of administration, peptides used, doses and duration of treatment tested all influence the outcomes of these studies.The current study also showed that MC2R mRNA expression was increased by ACTH at 12 and 24 h post-incubation in H295R cells (Supplementary Information).The low response to ACTH has been a drawback of H295R cells, which was attributed to low levels of MC2R in these cells 31 .In agreement with current findings, other studies showed that H295R cells showed higher basal MC2R expression levels and greater response to ACTH stimulation at time points above 12 h 32 .Another interesting piece of evidence from these results is the inhibitory effect of NLP and NESF-1 on endogenous NUCB mRNA expression, while ACTH stimulated NUCB1 and NUCB2 in human adrenal cortex cells.This effect of NLP and NESF-1 are likely due to a negative feedback on endogenous NUCBs due to the excess levels of processed peptides sensed by the cells during the in vitro treatment with synthetic peptides.
NLP and NESF-1 act through PKA/CREB mediated pathway to stimulate POMC synthesis in mouse corticotrophs 1 .Similarly, ACTH stimulates the production of GCs through MC2R-cAMP/PKA/CREB pathway in the adrenal cortex cell line, including Y1 mouse adrenocortical cells 33 .We hypothesized that NLP and NESF-1 modulate cortisol synthesis through the CREB-mediated pathway in human adrenal cortex cells.To assess the potential signalling pathway that mediates the inhibitory effect of NLP and NESF-1 on cortisol synthesis, the phosphorylation of CREB at different time points was first checked.In the current study, these peptides decreased P-CREB/T-CREB ratio at 90 min post-incubation.In the next step, we found that when H295R cells were preincubated with NLP and NESF-1, the PKA/CREB signalling-mediated cortisol secretion by forskolin was significantly decreased.These results demonstrate that AC/PKA/CREB pathway is critical to mediate the effect of NUCB-derived peptides on cortisol synthesis in human adrenal cortex cells.One report showed that NESF-1 stimulated intracellular calcium influx in hypothalamic neurons of rats, and this effect was abolished by pertussis toxin, which suggests that NESF-1 interacts with G-protein coupled receptor [GPCR] in brain cells 34 .CF568-labeled-NESF-1 and CF568-labeled-NLP bind to the membrane of GH3 cells, suggesting a possible GPCR-mediated action of NESF-1 and NLP in these cells 28 .A putative nesfatin-1 receptor GPCR12-like immunoreactivity was also detected in the pituitary and interrenal cells of goldfish 2 .As a result, these results and previous findings suggest the presence of a functional receptor on the surface of stress-related cells for NESF-1 and NLP.
We also studied the effect of IP injection of NLP on the genes as mentioned above in adrenal gland and pituitary of WT mice.There have been several studies on the administration of NESF-1 in animal models and its effect on either stress-related hormone synthesis and gene expression patterns or the emergence of anxiety-like behaviour.Repeated IP injection of NESF-1 induced anxiety-like behaviour in rats 35 .The increased anxiety-like behaviour was associated with elevated NUCB2/NESF-1 in the hippocampus, plasma and stomach of rats following the sequential stress induction 36 .IP and ICV administration of NESF-1 increased cortisol release and hypothalamic crh mRNA levels in goldfish, respectively 2 .However, in the current study, IP injection of NLP decreased the expression of steroidogenic enzymes [in agreement with the in vitro results], while increasing the expression of Pomc and Pcsk1 mRNA levels in mice pituitary.The level of endogenous Nucb1 and Nucb2 were decreased in the adrenal gland, and pituitary samples of NLP injected mice.These results show that NLP and NESF-1 regulate cortisol synthesis and steroidogenesis through ACTH-dependant and independent manner, respectively.
The genetic disruption of Nucb1 did not affect the genes mentioned above except for the downregulation of Hsd3β in the adrenal gland of female Nucb1 KO and Pcsk1 in the pituitary of male Nucb1 KO mice.These results were linked with tissue-specific upregulation of Nucb2 in the adrenal gland, while no changes were observed in the pituitary of male and female Nucb1 KO mice.The results of this study demonstrate that the expression of most steroidogenic enzymes remained unaffected in Nucb1 disrupted mice.This is possibly due to the compensatory upregulation of Nucb2 in the adrenal gland, which was detected in current research.The stress -related gene expression pattern in Nucb1 KO mice appears to be sex-and tissue-specific.Previous studies emphasized the predominant implication of NUCB2/NESF-1 in the mediation of anxiety in a sex-dependent manner.One study showed that circulating NESF-1 level in female patients was significantly correlated with the increased www.nature.com/scientificreports/severity of stress, while no correlation was observed in male patients 37 .Another study depicted the sex-dependant regulation of NUCB2/NESF-1 under depressive conditions with the positive correlation of circulating NESF-1 with an increased degree of depression in obese female patients.In contrast, such correlation was not observed in male patients 38 .Since the H295R cells is originated from a female patient, the pattern of mRNAs encoding steroidogenic enzymes and endogenous Nucbs found in this study can be interpreted as a female-specific effect.Since the endogenous hormonal milieu is absent, this in vitro approach has limitations.Male cells and in vivo studies using both males and females are crucial to confirm the conclusions reached here.In summary, this study provides the first evidence on the direct and indirect effects of NLP and NESF-1 on cortisol synthesis and steroidogenesis, and how genetic disruption of Nucb1 versus the injection of NLP affects HPA-related gene expression.The results of current study show that NLP and NESF-1 directly inhibit GC synthesis in an ACTH-independent manner.This report, along with previous publications expand the current understanding of NLP and NESF-1 as stress-responsive peptides and their role in the regulation of stress hormones synthesis.More studies on stress in response to NESF-1/NLP administration in different species are warranted.The role of NLP on the stress/HPA axis in humans is poorly understood.The implications of the results reported here on human diseases including anxiety and depression warrant further consideration.The outcome of this study and other studies set the stage to further explore NUCB peptides present within them in stress and anxiety in animals.They also raise possibilities for future studies aimed at its use in animals and humans as a biomarker or as a target for stress-related disorders.

Cell culture
Human adrenal cortex cell line [H295R, CRL-2128™] was purchased from ATCC.This cell line was derived from the NCI-H295 pluripotent adrenocortical carcinoma cells [ATCC CRL-10296].The cells were grown as per the protocols provided by the supplier in 10 cm cell culture dish containing DMEM:F12 Medium [cat no.30-2006, ATCC] supplemented with 1% ITS + Premix [cat no.354352, Corning, Canada] and 2.5% Nu-serum [cat no.355100, Corning, Canada], at 37 °C in 5% CO2 and 95% humidified atmosphere.When cells reached confluency, they were split at the desired density and used for specific experiments.

Peptides
Rat NES-1 (Cat no# 003-22B) was purchased from (Phoenix Pharmaceuticals INC. CA, USA).Murine NLP (> 95% purity) was custom synthesized (ABGENT, USA).The sequence of NESF-1 is: H-vpidvdktkvhnvepvesarieppdtglyydeylkqvievletdphfreklqkadieeirsgrlsqeldlvshkvrtrldel and NLP is: H-VPVDRAAPPQEDSQATETP-DTGLY YHRYLQEVINVLETDGHFREKLQAANAEDIKSGKLSQELDFVSHNVRT KLDEL.The putative bioactive core of NESF-1 and NLP are very highly conserved across human and rodent peptides, and our preliminary results found equipotent activity for these peptides in human cells.

Cortisol measurement
Cortisol was measured in cell lysate and cell culture media.For this purpose, H295R cells were seeded in 6 well plates and treated with NLP/NESF-1/ACTH [10 nM for peptides and 100 nM for the positive control group] for different time points, including 6 h, 12 h and 24 h.Then, cells were washed with cold PBS and trypsinized.After centrifugation at 15,000 g at 4 °C, trypsin-EDTA was removed, and cells were suspended in cold PBS and sonicated 3 times for 1-2 min.Cell culture media from all groups were collected to measure the secreted cortisol level in the media using human cortisol ELISA kit [cat no.COR31-K01, Eagle Bioscience, Inc, USA] according to manufacturer's instructions.All cell lysate samples were diluted with cold PBS.The assay sensitivity and dynamic range test were 0.4 μg/dL and 0.5-60 μg/dL, respectively.

Mechanism of action of NLP and NESF-1 on H295R cells
We reported that NLP and NESF-1 act through AC/PKA/CREB pathway to regulate POMC synthesis in mouse corticotrophs 1 .ACTH stimulates the production of GCs through MC2R-cAMP/PKA/CREB pathway in the adrenal cortex cell line, including Y1 mouse adrenocortical cells 32 .Therefore, we hypothesized that NLP and NESF-1 modulate cortisol synthesis through the CREB-mediated pathway in human adrenal cortex cells.To determine the cells signalling mediators, H295R cells were incubated with fresh media [control group] or NLP/ NESF-1 [experimental groups] at effective doses [10 nM for peptides and 100 nM for ACTH] for 90 min, and then phosphorylated [P]-CREB/total [T]-CREB ratio was assessed in the cell lysate.
In the second part of this study, H295R cells were preincubated with NLP or NESF-1 to block the CREBmediated pathway for 12 h, then forskolin [cat no.11018-5, Cayman Chemical Co, USA] as a specific adenylate cyclase stimulator [10 µM] was added to the media for 12 h.The cortisol level in cell lysate or cell culture media was measured using a cortisol ELISA kit as explained below.The dissolved forskolin in DMSO was less than Vol:.(1234567890

Effects of NLP and NESF-1 on steroidogenic enzymes and endogenous Nucb mRNAs in H295R cells
H295R cells at a confluency of 90% were treated with mouse NLP or rat NESF- , USA] at 100 nM was used as a positive control group.After the incubation period, cells were harvested, and the abundance of mRNAs for steroidogenic enzymes was assessed using qPCR.The abundance of endogenous Nucb mRNAs was also measured.The effective dose and time points were selected based on pilot studies [data not shown] and previous research 1 .In our previous study, we assessed the identity and similarity percentage between rat and mice peptides.Interestingly, rat and mouse NES-1 have 97.6% identity and 98.8% similarity in amino acid sequence.In addition, rat and mouse NLP share 97.4% identity and 98.7% similarity in amino acid sequence 1 .

Intraperitoneal NLP injection in wildtype mice and Nucb1 knockout [KO] mice
For the first study, C57BL/6J wildtype [WT] mice [n = 6 mice/group; both males and females; based on power analysis and reduced number of animals based on animal care approval] were used to study whether intraperitoneal [IP] injection of NLP affects HPA-related hormones and genes.All mice were fasted for 4 h [from 9 am] before the commencement of experiment.Mouse NLP was dissolved in sterile saline [0.9% NaCl] and then diluted to 100 µg/kg BW.It was administered to mice using insulin syringes [BD®, cat no.324911] attached to a 27G needle in the lower right quadrant of abdomen to avoid damage to internal organs.The selected dose was validated to be metabolically active and decrease food intake and modulate body weight [14].After 30 min, mice were euthanized, and blood samples and internal organs were collected.Age-matched mice were chosen and fed a standard rodent chow diet [cat no.500I, LabDiet, 7.94% carbohydrate, 28.67% protein, 13.38% fat, Energy density = 4.09 kcal/g].Mice were anesthetized using 3% isoflurane inhalation and were euthanized by cervical dislocation.Different tissues, including the adrenal gland and pituitary, from these mice were collected after cervical dislocation, total RNA was extracted, and the expression of stress-related genes was assessed in the cell lysate.

Total RNA extraction, cDNA synthesis, and real-time quantitative PCR
This section was carried out as described in previously reported protocols [1].Briefly, RNA was extracted using Ribozol [cat no.N580, VWR, USA], and then RNA's quantity and quality were determined by NanoDrop 2000 [Thermo Fisher Scientific].The RNA was reverse transcribed to cDNA using iScript Reverse Transcription Supermix for RT-qPCR [cat no.170884, Bio-Rad, USA] followed by the quantitative measurement of mRNA expression by qPCR in a CFX Connect Optic module [Bio-Rad, USA] following the requirements of the MIQE guidelines [17] and using SensiFAST™ SYBR No-ROX MIX [cat no.BIO-98050, Bioline, UK].The primers sequences and annealing temperatures are listed in Table S1 (Supplementary Information) purchased from Integrated DNA Technologies [IDT].Three negative controls, including no template DNA [NTC control], no reverse transcriptase control from cDNA synthesis process [RTC control], and a nuclease-free water sample [PCR control], were also included for each gene expression study.Thermal cycling set-up for all genes was the following: denaturation [95 °C for 5 s], annealing [specific for each primer for 25 s] and elongation [60 °C for 25 s], 35 cycles.At least 3 independent experiments with triplicates for in vitro studies [final samples ≥ 9] and more than 6 mice/group/ sex were considered.The abundance of mRNA was calculated based on the Pfaffl method and gene-specific efficiencies [18], relative to the geometric means of the 2 housekeeping genes.

Western blot analysis
The western blot protocol followed was described previously 1 .Briefly, total protein was extracted using RIPA Lysis buffer [cat no.89901, Thermo Fisher Scientific, USA] and the protein concentration was determined by Bradford assay.An equal amount of crude protein [40 μg] was electrophoresed on 8-16% Mini-Protean TGX gels [cat no.456-1104, Bio-Rad, USA], and the protein bands were transferred to nitrocellulose membranes [cat no.1704158, Bio-Rad, USA] using the Trans-Blot Turbo Transfer System [Bio-Rad, USA].After blocking the membranes, they were incubated with primary antibodies overnight at 4 °C followed by washing steps, incubation with secondary antibodies for 1 h and final washing.

Figure 1 .
Figure 1.H295R cells are immunoreactive for NUCB1/NLP (A) and NUCB2/NESF-1 (B), both shown in green color.DAPI stained DNA in blue color group.No primary antibody is negative control group (C).Arrows point to magnified cells shown on the top right inset.

Figure 2 .
Figure 2. NLP and NESF-1 decreased cellular cortisol content in H295R cells.The results presented are pooled from 3 independent studies with at least triplicates for each treatment.The same alphabets show no significant difference between groups, while different letters indicate significant differences among groups for each time point.

Figure 3 .
Figure 3. NLP and NESF-1 decreased the phosphorylation of CREB in H295R cells (A) and regulated cortisol release through a CREB mediated pathway (B).The results presented are pooled from 3 independent studies with duplicate/triplicate for each treatment.Same alphabets show no significant difference between groups, while different letters indicate significant differences among groups.Full size western gel images that served as the source for this figure are shown in Supplementary Fig. 2. Each of the image came from a single source gel image.

Figure 4 .Figure 5 .
Figure 4. NLP and NESF-1 affected the expression of steroidogenic enzyme mRNA abundance in H295R cells.The results presented are pooled from 3 independent studies with at least triplicates for each treatment.Asterisks show statistical differences between experimental and control groups.

Figure 6 .
Figure 6.IP injection of 100 µg/Kg BW NLP affected the expression of steroidogenic enzymes, endogenous Nucb and Mc2r in the mouse adrenal gland.Studies were done on 6-7 tissue samples of mixed-sex C57BL/6 mice.Asterisks show a significant difference between the experimental and control groups.

Figure 7 .
Figure 7. IP injection of 100 µg/Kg BW NLP affected the stress-related genes and endogenous Nucb in the mouse pituitary.Studies were done on 6-7 tissue samples of mixed-sex C57BL/6 mice.Asterisks show a significant difference between the experimental and control groups. https://doi.org/10.1038/s41598-024-54758-7 https://doi.org/10.1038/s41598-024-54758-7 To characterize the effects of disruption of Nucb1 in the second study, breeding pairs of homozygous C57BL/6NCrl-Nucb1em1[IMPC]Mbp/Mmucd mice (global Nucb1 disrupted mice) were purchased from the University of California [generated by Dr. Kent Lloyd, Mouse Biology Program, University of California-Davis, USA].These breeding pairs were used to establish a colony of homozygous Nucb1 disrupted mice.In these mice, the exon 4 and flanking splicing regions of Nucb1 were constitutively deleted using CRISPR Cas9 gene editing technology in C57BL/6J mouse zygotes.All the animals were housed under 12 h light:12 h dark cycle [7 am-7 pm], humidity [30-60%], and temperature [18-22 °C] controlled vivarium located in the College of Medicine Lab Animal Services Unit, University of Saskatchewan.All protocols were prepared based on guidelines of the Canadian Council for Animal Care and were approved by the University of Saskatchewan Animal Care Committee [2012-0033].The experiments conducted adhered to the ARRIVE guidelines on animal use and care.
Statistical analysis was conducted using the SPSS statistical software [IBM SPSS Statistics for Windows, Version 23.0, USA].The normality of data distribution was analyzed by Shapiro-Wilk's test and all data were normally distributed.The homogeneity of variances was checked by Levene's test.The significance level was set at p < 0.05.The single comparison was performed by Student's t-test, and multiple comparisons were performed by one-way ANOVA followed by Tukey's multiple comparison test.All graphs were plotted by GraphPad Prism [GraphPad Software, Inc., Prism 8 for Windows, Version 8.4.2,USA].

Table 1 .
A comparison of mRNAs in the adrenal gland of wildtype (WT) vs Nucb1 KO mice.Studies were conducted on 6 tissue samples/mice per group.No change, an increase, or a decrease in the knockout male or female mice when compared to their respective control groups is denoted in the tables.
.1% in cell culture media.The concentration and time point for forskolin incubation were chosen based on the recommended dose range in the supplier catalogue and were independently validated in pilot studies [data not shown].ACTH treated cells were used as a positive control group.